E gene, 95% CI −0.938 to −0.834) and −0.914 , respectively. The limit of detection of the MARK-B was measured using the viral culture reference samples and found to be 1 x 102 pfu/mL. The magnetic force-assisted electrochemical immunoassay-based Ag test can be used to rapidly detect SARS-CoV-2 infections, and the corresponding fully automated portable device can provide easy readability and semi-quantitative results.Rapid antigen (Ag) tests for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provide quick results, do not require specialized technical skills or infrastructure, and can be used as a point-of-care method to prevent the spread of coronavirus disease (COVID-19). The performance of a magnetic force-assisted electrochemical immunoassay-based test, namely the MARK-B COVID-19 Ag test , was evaluated using 170 nasopharyngeal swab specimens and compared to that of RT-PCR and commercial rapid Ag test . The overall sensitivity and specificity of the MARK-B test were 90.0% (95% CI 79.4%–96.2%) and 99.0% (95% CI 95.0%–99.9%), respectively, with a kappa coefficient of 0.908. The correlations between the electrical current values of MARK-B and the Ct values of RT-PCR were −0.898 ( Reverse-transcription polymerase chain reaction (RT-PCR) is considered the standard method for the diagnosis of coronavirus disease COVID-19) because of its high sensitivity and specificity 9 because. HoweverThe MARK-B COVID-19 Ag test  is a portable point-of-care device based on magnetic force-assisted electrochemical immunoassay (MESIA) designed to detect SARS-CoV-2 nucleocapsid antigens. Notably, this newly developed immunoassay, MESIA, is reportedly suitable for measuring low concentrations of proteins for cancer detection and has the potential to detect other protein biomarkers , 21. MESNasopharyngeal swab (NP) specimens were collected from patients with COVID-19 symptoms. These patients were subjected to an RT-PCR test for SARS-CoV-2 by clinicians in Eunpyeong St. Mary’s Hospital or Incheon St. Mary’s Hospital, Republic of Korea, between October 2020 and February 2021. NP specimens were placed in a universal transport medium , and they were initially evaluated using RT-PCR. The remnant NP specimens were stored at −70°C, and they were used to evaluate the clinical performance of the MARK-B tests. Clinical information, such as symptoms and days from onset of symptoms (DFOS), was retrieved from medical records. This study was approved by the Institutional Review Board of the Catholic University of Korea, Republic of Korea (XC21DDDT0025), and the informed consent was waived.in vitro medical device that is based on principles of MESIA and intended for the qualitative and semi-quantitative detection of the SARS-CoV-2 nucleocapsid antigens. When a sample is loaded into a cartridge, it flows into the microchannel, containing gold-coated magnetic nanoparticles and an electrochemical sensor. The analytes form immune complexes under external magnetic fields. The targeted antigen level is measured based on the electrochemical redox signal of gold-coated magnetic nanoparticles conjugated with the antibodies. The electrical signal is measured using a fully automated portable device, the MARK-B 1 Analyzer (BBB), and the result is determined in 10 min based on the cut-off value that has been set as per the manufacturer’s instructions. The preset cut-off value is determined for each lot once manufactured based on the mean and the standard deviation of the electrical signal obtained from negative samples. The QR code on each cartridge contains the information and the analyzer can recognize the cut-off of each cartridge when the QR code is scanned. The assay provides a result that indicates the absence or presence of SARS-CoV-2, along with the quantity of the captured targets from the specimen, measured using the electrical signals.The MARK-B test is an The MARK-B tests using NP specimens were performed according to the manufacturer’s instructions, which recommended the direct use of a nasopharyngeal swab or an aliquot of UTM. The previously stored UTM samples were thawed at room temperature for 30 min and vortexed for 10 s. Manufacturer recommends using 400 μL of UTM to extract SARS-CoV-2 nucleocapsid proteins from the specimen by mixing it with the extraction buffer provided with the product. However, the remnant NP specimen after RT-PCR was not enough. Therefore, the volume of the extraction buffer added was 200 μL for MARK-B™ COVID-19 Ag, and the sample was 1:1 diluted after UTM is added. Three drops of the specimen were applied to the cartridge, and the device was subjected to operation. Test results above the lot-specific cut-off value  were considered positive.E and RdRp genes of SARS-CoV-2 were amplified over 40 cycles using the Applied Biosystems 7500 RT-PCR system . Samples were considered as SARS-CoV-2-positive when both targets of viral RNA had been amplified under a cycle threshold (Ct) of 38.0.Viral RNA extracted from the nasopharyngeal specimens (200 μL each) was performed using the NX-48 viral NA kit  and the Nextractor NX-48 system (Genolution). Nucleic acids were extracted according to the manufacturer’s instructions. SARS-CoV-2 was amplified using real-time RT-PCR, with a commercial Real-Q 2019-nCoV Detection Kit . The The STANDARD Q COVID-19 Ag Test  was used for the detection of SARS-CoV-2 antigens in NP specimens. Notably, the STANDARD Q COVID-19 Ag (SDQ) and the MARK-B tests were conducted concurrently. The stored UTM samples were thawed at room temperature for 30 min and then vortexed for 10 s. The manufacturer recommends using 350 μL of UTM and 350 μL of extraction buffer for the dilution factor 1:1. As described earlier, the remnant NP specimens was not enough, 200 μL of the NP specimen from stored UTM was mixed with the extraction buffer provided in the test kit (1:1). Three drops of the extracted specimen were applied to the test device, and the result was recorded within 15–30 min. The test result was considered positive when both the control and the test lines were colored.5 pfu/ml and samples were serially diluted to 1.0 x 105 pfu/ml, 1 x 104 pfu/ml, 1 x 103 pfu/ml, 4 x 102 pfu/ml, 2 x 102 pfu/ml, and 1 x 102 pfu/ml. MARK-B and SDQ tests were repeated five times for each diluted sample according to the manufacturer’s instructions. Cell culture procedures were performed according to biosafety level 3 (BSL-3) conditions.To compare the limit of detection (LOD) between two rapid Ag kits, a serial dilution of SARS-CoV-2 samples were used. Vero E6 cells  were cultured and incubated with the SARS-CoV-2 strain . The viral concentration was quantified to be equivalent to 6.5 x 10E and RdRp genes obtained via RT-PCR and the measured MARK-B electrical current values was evaluated using Pearson’s correlation coefficient (r). Receiver operating characteristic (ROC) curve analysis was performed to evaluate the MARK-B lot-specific cut-off value with clinical samples. Cohen’s kappa coefficient (k) was used to assess the inter-rater reliability among SARS-CoV-2 antigen assays. Notably, k < 0 indicated no agreement, while agreement was considered to be slight for k = 0–0.20, fair for k = 0.21–0.40, moderate for k = 0.41–0.60, and substantial for k = 0.61–0.80; k = 0.81–1 represented an almost perfect agreement. The statistical significance threshold was set at P < 0.05.Statistical analysis was performed using the MedCalc software ver 19.6.1 . Figures were created using GraphPad Prism ver 9.1.2 . The Kolmogorov–Smirnov test was used to assess the normality of the distribution. The correlation between the Ct values of the In total, 60 samples were analyzed using RT-PCR and were considered SARS-CoV-2-positive, while 110 samples were considered SARS-CoV-2-negative. Among the 60 samples that were confirmed as positive, 24 NP specimens had been submitted 0–3 DFOS, 24 NP specimens had been collected 4–7 DFOS, and 12 had been obtained >8 DFOS.k value of MARK-B compared to RT-PCR was 0.908. Based on the RdRp Ct value ranges , and that between the Ct values of the RdRp gene and the MARK-B values revealed a value of r = −0.914  were significantly higher , that of MARK-B-positive/SDQ-weak positive was 22.4 (SD 1.6), that of MARK-B-positive/SDQ-negative was 26.4 (SD 2.5), and that of MARK-B-negative/SDQ-negative was 31.8 (SD1.1)  and 100% specificity (95% CI 96.7%–100%) for SARS-CoV-2 Ag detection. There were 26 false negatives, and the CR tests . The sen (SD1.1) . There wr groups .2 pfu/mL, and SDQ was 1.0 x 104 pfu/mL, respectively . Additionally, it was characterized by 95.9% overall agreement and a 0.908 k value, indicating an extremely good match between the results of RT-PCR and the MARK-B test. Both the commercial and the MARK-B Ag tests exhibited higher sensitivity for Ct values <25, and the sensitivity decreased as Ct values increased. The SDQ test reportedly exhibits a sensitivity above 95% for Ct values <25 and 53.9–62.1% for Ct value >25 [In the present study, the performance of the MESIA-based Ag test MARK-B was evaluated using clinical samples and compared with the performance of RT-PCR and commercial Ag tests. The MARK-B tests displayed 90.0% sensitivity and 99.0% specificity across the samples. Moreover, the MARK-B test exhibited 100% sensitivity for samples whose RT-PCR results were Ct < 25 and 84.6% sensitivity for both alue >25 , 22–25. alue >25 , 25. Allalue >25 ; thus, malue >25 .2 times lower viral concentrations than SDQ.The MARK-B tests are automatically conducted using a portable device analyzer that regulates the magnetic field and measures electrochemical signals. The qualitative results were determined based on a cut-off value, and the measured electrochemical signals were also presented. The ROC curve analysis with clinical samples demonstrated that the lot-specific cut-off value was appropriately established. The use of visual readout rapid Ag tests can produce ambiguous results at low antigen concentrations, while instrument-based Ag tests yield results with clarity. For instance, when interpreting the visual readout Ag test, the presence of a line, regardless of the faintness of the line, indicates a positive result. There were 10 weak-positive cases within the SDQ results and 20 cases of MARK-B-positive/SDQ-negative results, and there were significant differences between these sample groups. Notably, the MARK-B device provided readability for clinical samples with higher Ct values, and the MESIA technique improved the sensitivity of the immunoassay for detecting SARS-CoV-2 antigens. The LOD measurement of two rapid Ag tests were conducted to verify the sensitivity differences between the rapid Ag tests using viral culture reference samples. In line with tests using clinical samples, MARK-B tests showed a LOD at approximately 10Furthermore, the electrical current values of MARK-B and the Ct values of RT-PCR were highly correlated. We plotted log2(current) values vs. Ct values and fitted with a line. While the signal was saturated at the concentrations higher than Ct 20, the log2(current) vs. Ct showed a linear relationship in the range from Ct 20 to Ct 33, as shown In conclusion, the MARK-B test, a MESIA-based rapid Ag test, showed higher sensitivity compared to commercial rapid Ag tests for the detection of SARS-CoV-2. Furthermore, the MESIA technique and automated portable device provided results with improved clarity in 15 min as well as reliable semi-quantitative measurement. These results indicate that these rapid Ag tests can be useful for preventing the spread of COVID-19 via timely diagnosis and subsequent containment measures. 3 Aug 2021PONE-D-21-20548Evaluation of the clinical performance of a magnetic force-assisted electrochemical immunoassay for the detection of SARS-CoV-2 antigensPLOS ONEDear Dr. Lee,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Please describe the limitation of your present study, which was commented by one of the reviewers.plosone@plos.org. 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For instructions see: We look forward to receiving your revised manuscript.Kind regards,Etsuro ItoAcademic EditorPLOS ONEJournal Requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf" ext-link-type="uri" xlink:type="simple">https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/"https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/2. Please upload a new copy of Figure 1 as the detail is not clear. Please follow the link for more information: 3. Please expand the acronym “BBB”  so that it states the name of your funders in full.This information should be included in your cover letter; we will change the online submission form on your behalf.Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: PartlyReviewer #3: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: NoReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The Authors evaluated the MARK-B COVID-19 Ag test  that is a portable point-of-care device based on the use of a magnetic force-assisted electrochemical immunoassay (MESIA) designed to detect SARS-CoV-2 antigens. Notably, this newly developed immunoassay, MESIA, reportedly suitable for measuring low concentrations of proteins for cancer detection and with the potential to detect any other biomarkers.The evaluation was done against RT PCR as the gold standard for SARS COV-2 detection and another immunochromatographic manual antigen assay for SARS CoV-2, showing a significant higher correlation with RT PCR and a significant higher sensitivity over the standard antigen assay. Overall, this data confirms that MESIA may provide enhanced sensitivity for point-of-care devices that involve the detection of SARS-CoV-2.The study is well conducted, each section is well written and developed, statistical measures correctly applied. There is one general limitation in this study:https://www.finddx.org/covid-19/pipeline/?section=immunoassays#diag_tabThe MARK-B test has not been CE/FDA-cleared or approved so far for SARS CoV-2, but these validations are pending. Therefore, this is a clear limitation of the study conducted by the Authors, in terms of extension and commercialization of this device. The Authors should discuss this issue and clarify if there are any on-going news on this important issue which is essential for the scientific community. Moreover, the test is not cited by FIND as the date of today July 15 among the 653 antigen immunoassays for SARS CoV-2 enlisted in the web site Is this just for Asia? The Authors must clarify it.Minor issuesMaterial and Methods:The semi-quantitative/quantitation system used by MESIA for their electric signal should be specified by the Authors as they use it in the Figures, as well as the nature of the cut-off value and the type of antigen detected by this system.Reviewer #2: This paper describes the evaluation of a magnetic force-assisted electrochemical immunoassay -based test compared to other commercial rapid Ag tests by application to clinical samples. Authors have successfully compared the results provided by this device with those from other methodologies applying them to a large number of samples. In my opinion, the information offered in the article is very interesting, the electrochemical sensor has demonstrated a good behavior, but the experimental work and the new data in the article are not relevant from the point of view of scientific research. I consider that the paper should not be published in its current state in PLOs One.Reviewer #3: This is a clear well-written paper describing the performance of a new Ag test for diagnosis of COVID-19 infection. As an independent of assessment of the test, the publication provides important data to users who may be considering use of the test. I recommend publication after revision to address some minor weaknesses in the paper:1. The main weakness is associated with the comparison to an existing commercial Ag test and the interpretation of the comparisona. In the abstract and main text their are locations where the authors describe their paper as comparing their test to commercial antigen tests. These statements should be clarified to indicate that they compared to a (as in one) antigen test, and isn't necessarily reflective of how the test would perform to the range of available tests.b. Some discussion is warranted regarding the criteria for the selection of the comparator test, and the relative performance of this test. Relative to the many Ag tests that are now available commercially, does the published data for the SDQ test place it at the top, middle or bottom of the range for clinical sensitivity and accuracy?c. It would be helpful to provide some testing with a reference sample  that could allow direct comparison of the analytical performance of the antigen assays to each other  and determination of whether the clinical performance is a direct reflection of the analytical performance).2. The volume of UTM used to extract the sample should be provided and, if applicable, a discussion of how the volume of UTM may differ from the volume of extraction buffer used in the standard swab protocols for the MESIA and SDQ tests, and how that may affect performance.3. Ct should be proportional to Log2(RNA concentration) since it represents the number of RNA doubling steps required to achieve a detectable PCR signal. It is therefore probably most appropriate to plot Log2(MESIA current) vs. Ct, which should provide a slope approaching 1. If the log(current) vs. Ct is not linear, that should be discussed.4. In the methods section, it isn't very clear how the MESIA technique works. I think readers would provide a little more detail or, alternatively, a reference to where they could go for more details.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  13 Sep 2021.AttachmentResponse_to_Reviewers.docxSubmitted filename: Click here for additional data file. 27 Sep 2021Evaluation of the clinical performance of a magnetic force-assisted electrochemical immunoassay for the detection of SARS-CoV-2 antigensPONE-D-21-20548R1Dear Dr. Lee,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Etsuro ItoAcademic EditorPLOS ONEReviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #3: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #3: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #3: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #3: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #3: Some minor comments/suggestions based on the response to my original review:1. The description of the assay technology in your response to the reviewers comments is much clearer than in the edits to the manuscript. I suggest just using the text from the response in the manuscript.2. The line in the log2 antigen vs. Ct count plot should be calculated using the log2 transformed values and should look like a straight line in the plot. Interestingly, the slope of that line will be about -0.3 indicating that an 8-fold change in nucleic acid levels (3 Ct counts) corresponds to roughly a 2-fold change in the antigen assay signal (roughly a cube root dependence). The table of assay signals for the virus titration also shows roughly a cube root dependence.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #3: No 29 Sep 2021PONE-D-21-20548R1 Evaluation of the clinical performance of a magnetic force-assisted electrochemical immunoassay for the detection of SARS-CoV-2 antigens Dear Dr. Lee:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProf. Etsuro Ito Academic EditorPLOS ONE


The current study aimed to explore the α-blockers’ anti-QS activities. Within comprehensive in silico investigation, the binding affinities of seven α-adrenoreceptor blockers were evaluated towards structurally different QS receptors. From the best docked α-blockers into QS receptors, terazosin was nominated to be subjected for further in vivo and in vitro anti-QS and anti-virulence activities against Chromobacterium violaceum and Pseudomonas aeruginosa. Terazosin showed a significant ability to diminish the QS-controlled pigment production in C. violaceum. Moreover, Terazosin decreased the P. aeruginosa biofilm formation and down-regulated its QS-encoding genes. Terazosin protected mice from the P. aeruginosa pathogenesis. In conclusion, α-adrenergic blockers are proposed as promising anti-virulence agents as they hinder QS receptors and inhibit bacterial espionage.Antimicrobial resistance is among the world’s most urgent public health problems. Diminishing of the virulence of bacteria is a promising approach to decrease the development of bacterial resistance. Quorum sensing (QS) systems orchestrate the bacterial virulence in inducer–receptors manner. Bacteria can spy on the cells of the host by sensing adrenergic hormones and other neurotransmitters, and in turn, these neurotransmitters can induce bacterial pathogenesis. In this direction,  Drug repurposing is discovering new applications of the well-known drugs which are known for specific clinical use. Re-evaluating the medical use of already-permitted safe drugs has gained increasing interest as a promising strategy that acquires different merits. These advantages include saving time and costs to carry out further pharmaceutical, pharmacological, and toxicological studies for already-approved safe drugs ,2,3. TheTreating antimicrobial-resistant infections or discovering new antimicrobials are among the most valuable aims of drug repurposing which has aroused the curiosity of several research groups ,13,14,15α-blockers as anti-QS and anti-virulence agents.Bacteria hire a complicated system of inducers and receptors to communicate with each other, named quorum sensing (QS) ,35. Profα-blockers, hinders the bacterial receptor-based sensing and mitigates the bacterial virulence 3]. To avvaluated ,30. BrieC. violaceum was evaluated as previously described [C. violaceum suspensions (O.D600 1) in microtiter plates and incubated at room temperature overnight. Then, plates are left to dry and the violacein pigment was extracted with dimethylsulfoxide (DMSO) and the absorbances were detected at 590 nm. The results were calculated as a percentage change from the untreated negative control cultures.The terazosin ability to diminish the QS-controlled violacein production by escribed ,30. BrieP. aeruginosa, treated or untreated with terazosin at sub-MIC, were extracted using the provided protocol by the Purification Kit Gene JET RNA . The expression of P. aeruginosa QS-encoding genes pqsA, pqsR, rhlI, rhlR, lasI, and lasR, as well as PmrAB system-encoding genes pmrA and pmrB, were quantified in the presence and absence of terazosin at sub-MIC by qRT-PCR. The relative levels of genes’ expressions were normalized to the ropD expression level as a housekeeping gene. The used primers were listed in  100 µL LB broth aliquots in the absence or presence of terazosin at-sub-MIC were transferred to microtiter plates and incubated at 37 °C for 24 h. Then, planktonic cells were washed out, and adhered cells were fixed with 99% methanol for 20 min and stained with 1% crystal violet for 25 min. Excess dye was washed out and plates were left to dry. Crystal violet was extracted with 33% glacial acetic acid and the absorbances were detected at 590 nm. The biofilm formation was considered as a percentage change from untreated cultures. To visualize the biofilms’ formation, the biofilms were left to be formed on cove slips in the absence or presence of terazosin at sub-MIC.The crystal violet method was used to assay the effect of terazosin at-sub-MIC on the biofilm production in escribed ,106. P. P. aeruginosa swimming or swarming was evaluated in the absence or presence of terazosin at sub-MIC as described earlier [P. aeruginosa inoculums (1 × 106 CFU/mL) were centrally inoculated in 0.3% or 0.5% LB agar plates provided with or without terazosin at sub-MIC for swimming or swarming motility assay, respectively. After overnight incubation, the zones of motility were measured in mm.The  earlier ,30. BrieP. aeruginosa cultures treated or not with terazosin at sub-MIC. Equal volumes of supernatants were mixed with fresh 2% erythrocytes suspensions, incubated at 37 °C for 2 h, and centrifuged. At the same conditions, negative control of un-hemolyzed erythrocytes and positive control were prepared by adding 0.1% sodium dodecyl sulfate (SDS) to an erythrocyte suspension. The absorbances were detected at 540 nm, and the hemolysis in the presence or absence of terazosin was compared to positive and negative controls. The results of treated P. aeruginosa were evaluated as a percentage change from the hemolysis of untreated control cultures.The anti-hemolytic activity of terazosin at sub-MIC was evaluated as previously described ,23. BrieP. aeruginosa was evaluated using the skim milk agar method [P. aeruginosa cultures treated or not with terazosin at sub-MIC. Equal volumes of supernatants collected from treated or untreated P. aeruginosa were moved to the wells in 5% skim milk agar plates and incubated at 37 °C overnight. The diameters of clear zones due to protease were measured in mm. The results obtained by treated P. aeruginosa were evaluated as a percentage change from those obtained by untreated bacterial controls.The anti-proteolytic activity terazosin at sub-MIC against r method ,24. BrieP. aeruginosa was assessed using the Congo red method [P. aeruginosa cultures treated or not with terazosin at sub-MIC. Supernatants were mixed with the ECR reagent, incubated 37 °C for 6 h, and then centrifuged to remove insoluble reagent. The absorbances were measured at 495 nm. The elastolytic activity of terazosin-treated cultures was evaluated as percentage change from untreated bacterial culture.The anti-elastolytic activity of terazosin at sub-MIC against d method ,30. The P. aeruginosa pyocyanin pigment was assessed as described earlier [P. aeruginosa overnight cultures were inoculated in 1 mL of LB broth with or without terazosin at sub-MIC, incubated at 37 °C for 48 h, and centrifuged. The absorbances of the produced pyocyanin were measured at 691 nm. The terazosin inhibitory effect on the production of pyocyanin was calculated as a percentage change from untreated bacterial culture.The inhibitory effect of terazosin at sub-MIC on formation of the  earlier ,49. BrieP. aeruginosa tolerance to oxidative stress [P. aeruginosa cultures were spread uniformly on the surface of LB agar plates, provided with or without terazosin at sub-MIC. Then, 10 μL of hydrogen peroxide (1.5%) was added on the sterile paper disks placed on the prepared LB agar plates. After overnight culture at 37 °C, the inhibition zones of P. aeruginosa, treated or untreated with terazosin, were measured in mm.The disk assay method was employed to evaluate the effect of terazosin at sub-MIC on the e stress . BrieflyP. aeruginosa, as previously described [P. aeruginosa cultures in LB broth, with or not terazosin at sub-MIC, were adjusted to approximately 1 × 106 CFU/mL) in phosphate-buffered saline (PBS). Three-week-old female Mus musculus mice were distributed in 4 groups (n = 10). The test group was intraperitoneally (ip) injected with 100 μL of terazosin-treated P. aeruginosa in sterile PBS. Two negative control groups were uninfected or were injected with sterile PBS. The positive control group was injected with untreated P. aeruginosa. The survival of mice was observed over 5 successive days and plotted using Kaplan–Meier method.To assess the in vivo anti-virulence activities of terazosin, the mice survival model was used to evaluate the terazosin’s in vivo protective activity against escribed ,49. Brieα-adrenergic blockers as anti-virulence agents. Based on the outcome of the in silico study, terazosin as a top dual QS affinity α-adrenoreceptor hit was identified, which is promising for further testing its anti-virulence activities. Terazosin significantly mitigated the QS-controlled virulence factors in C. violaceum and P. aeruginosa. Terazosin downregulated QS-encoding genes and protected mice against P. aeruginosa. This study shows the terazosin anti-virulence activity is owed to its anti-QS effects besides its blockade to bacterial espionage on the host cells and decreasing bacterial resistance to killing inside immune cells. In this work, the anti-virulence activities of α-adrenergic blockers were preliminary explored, thus keeping the door open for further detailed investigation prior to their clinical application.The development of bacterial resistance is an endless battle that mandates a dynamic development of new approaches and strategies. Repurposing safe and approved drugs to curtail bacterial virulence is one of the promising approaches and acquires multiple advantages. In this study, we aimed to repurpose


To compare the outcomes of drug-eluting bead bronchial arterial chemoembolization (DEB-BACE) with and without microwave ablation (MWA) for the treatment of advanced and standard treatment-refractory/ineligible non-small cell lung cancer (ASTRI-NSCLC).A total of 77 ASTRI-NSCLC patients who received DEB-BACE combined with MWA  or DEB-BACE alone  were included. Clinical outcomes were compared between groups A and B. Kaplan–Meier methods were used to compare the median progression-free survival (PFS) or overall survival (OS) between the two groups. Univariate and multivariate Cox proportional hazards analyses were used to investigate the predictors of OS for ASTRI-NSCLC treated with DEB-BACE.P = 0.002). The median PFS in groups A and B was 7.0 and 4.0 months, respectively, with a significant difference (P = 0.037). The median OS in groups A and B was both 8.0 months, with no significant difference (P = 0.318). The 6-month PFS and OS rates in groups A and B were 75.0% and 78.6%, 22.4% and 59.2%, respectively, while the 12-month PFS and OS rates in groups A and B were 17.9% and 28.6%, 14.3% and 22.4%, respectively. Of these, a significantly higher 6-month PFS rate was found in group A . The cycles of DEB-BACE/bronchial artery infusion chemotherapy  and postoperative immunotherapy  were identified as the predictors of OS in ASTRI-NSCLC treated with DEB-BACE.No severe adverse event was found in both groups. Pneumothorax was the predominant MWA-related complication in group A, with an incidence rate of 32.1% (9/28). Meanwhile, no significant difference was found in DEB-BACE-related complications between groups A and B. The overall disease control rate (DCR) was 61.0% (47/77), with a significantly higher DCR in group A (85.7% vs. 46.9%, MWA sequentially combined with DEB-BACE was superior to DEB-BACE alone in the local control of ASTRI-NSCLC. Although the combination therapy reveals a trend of prolonging the OS, long-term prognosis warrants an investigation with a longer follow-up. Primary lung cancer has an incidence and mortality of over 2.2 million and 1.79 million, respectively, estimated globally in 2020 . The staIn recent decades, computed tomography (CT)-guided microwave ablation (MWA) has been recommended as a primary treatment option by several guidelines , 8, espeThe optimal cutoff value of tumor diameter in predicting local recurrence after MWA was 3 cm, with the potential mechanisms for incomplete ablation in large tumors including the limited size and homogeneity of tumor necrosis achieved by MWA and the heat-sink effect caused by the blood vessels in ablated tumor . In theoThis single-center case-control study included all ASTRI-NSCLC patients who underwent DEB-BACE at this institution. The institutional ethics review board approved this study. The study protocol was conducted per the Declaration of Helsinki. Informed consent was waived due to the retrospective nature of this study. ASTRI-NSCLC patients who received DEB-BACE between May 2017 and April 2021 were allocated to the combination therapy group  and DEB-BACE alone group (group B). Informed consent of MWA or/and DEB-BACE was obtained before these procedures. Patient inclusion criteria were as follows: (a) age of 18–85 years; (b) advanced NSCLC that is ineligible for or is refractory to standard treatments; and (c) Eastern Cooperation Oncology Group score of 0–2. Patient exclusion criteria were as follows: (a) concomitant radioactive seed implantation was performed during MWA procedure; (b) incomplete data; (c) lost to follow-up; (d) MWA combined with bronchial artery infusion chemotherapy (BAI); and (e) period between MWA and DEB-BACE longer than 1.5 months.via previous percutaneous needle aspiration biopsy or surgery or fiberoptic bronchoscopy. Positron emission tomography or CT was performed to evaluate the tumor location, quantity, size, and local and distant metastases. The clinical TNM staging system of the Union for International Cancer Control (8th edition) was applied in identifying the tumor stage  guidelines . As descvia a femoral artery approach. A 5-French pigtail catheter  was initially used for aortic arch angiography to detect the origins of tumor-feeding arteries. Then, a 5-French cobra  or left gastric catheter  was used to select the bronchial arteries or non-bronchial systemic arteries. Super-selective catheterization with a 1.98-French microcatheter  was inserted coaxially. The chemotherapeutic regimen of paclitaxel  was administered during BAI for patients who had already received systemic platinum-based chemotherapy, while nedaplatin  was administered for patients who had not received systemic platinum-based chemotherapy. Gemcitabine  or endostatin  was infused on demand. BAI was followed by DEB-BACE, which was performed using 100–300 μm or 300–500 μm or 500–700 μm CalliSpheres microspheres  loaded with gemcitabine  or pirarubicin . The CalliSpheres microspheres were first mixed with gemcitabine or pirarubicin at a temperature of 23°C–28°C for 30 min and vibrated every 5 min. Then, iodixanol  was added at a 1:1 ratio. The DEB-BACE was performed slowly and carefully in tumor-feeding arteries under fluoroscopic monitoring to avoid reflux into nontarget vessels. The technical endpoint of embolization was the absence of additional tumor staining or stasis/near stasis of the tumor-feeding arteries. For patients who received combination therapy, DEB-BACE was performed 2–45 days after MWA, followed by repeated DEB-BACE/BAI on demand. For the patients who received a repeated DEB-BACE/BAI, DEB-BACE was performed if abundant tumor staining was revealed in angiography, while BAI alone was performed for those patients without.As described previously , DEB-BACShort-term follow-up with CT was conducted 1–5 days after MWA during hospitalization and 3–4 weeks after MWA at an outpatient visit to detect postprocedural complications, including pneumothorax and pleural effusion. Chest tube placement was performed for patients with moderate and severe pneumothorax or pleural effusion . ImmunotMWA- or DEB-BACE-related complications were evaluated according to criteria from the SIR , 23. EnhP-value <0.05 in the univariate analyses were entered as candidate variables into a stepwise Cox proportional hazards analyses. The results of the multivariate analyses indicated the predictors for the OS. A P-value <0.05 was considered to indicate statistical significance in the univariate and multivariate Cox proportional hazards analyses.Categorical variables are described as frequencies and percentages, and continuous variables are described as the mean ± SD. Statistical analyses were performed using SPSS 25.0 for Windows . The clinical characteristics, complications, and prognostic data were compared by Student’s t-test or Mann–Whitney U test for continuous variables and by chi-square test for categorical variables. Of these, Kaplan–Meier methods were used to compare the median PFS or OS between the two groups. The OS for ASTRI-NSCLC treated with DEB-BACE was estimated using the Kaplan–Meier method. The possible predictors of OS included 21 parameters on demographics, treatment history, MWA or/and DEB-BACE factors, and radiological features. Variables with a P = 0.021), whereas other variables revealed no differences. There were 21 patients (27.3%) who received immunotherapy after DEB-BACE, with PD-L1 blockade administered in six patients (7.8%) and PD-1 blockade administered in 15 patients (19.5%).A total of 77 NSCLC patients . There were nine patients (11.7%) who achieved PR at 3 months after MWA or/and DEB-BACE (P = 0.002).Detailed clinical outcomes between the two groups are presented in P = 0.001; P = 0.002; Detailed results of univariate and multivariate analyses are presented in The first-line therapeutic strategy for unresectable advanced NSCLC is chemoradiotherapy, which can provide an objective response rate (ORR) of 23%–34% and a median OS of 11.6 months after systemic platinum-based chemotherapy, or an ORR of 51.5%, a median OS of 22.0 months, and a median PFS of 17.0 months after chemoradiotherapy –27. The In recent decades, BACE/BAI has been reported as an effective and safe approach for advanced NSCLC, especially for chemoradiotherapy-ineligible/rejected patients, with a median PFS of 6.5–14.0 months and a median OS of 13.1–25.0 months –31. NSCLDEB microspheres can provide a higher local drug concentration while reducing the systemic drug concentration and toxicity . SeveralThe mechanisms of MWA are that microwaves can induce the oscillation of the water molecules that flip back and forth at a speed of 2–5 billion times a second depending on the frequency of the wave itself, allowing for a superior convection profile and causing coagulative necrosis conforming to the target sites . The optThis study has several limitations. First, it was a retrospective study; patient selection bias may exist. Second, the sample size was still limited for patients treated with combination therapy. Third, this study consists of advanced patients who are ineligible for or are refractory to standard treatments, and heterogeneity may exist. Fourth, the sequence of MWA and DEB-BACE is required to be further investigated. Fifth, the follow-up period was still limited, and the long-term evaluation of OS for combination therapy should be further explored.In conclusion, the combination therapy of MWA and DEB-BACE is an effective and safe approach for ASTRI-NSCLC. MWA sequentially combined with DEB-BACE was superior to DEB-BACE alone in the local control of ASTRI-NSCLC. Although the combination therapy reveals a trend of prolonging OS, long-term prognosis warrants an investigation with a longer follow-up.The data used to support the findings of this study are available from the corresponding author upon request.The studies involving human participants were reviewed and approved by Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences. The ethics committee waived the requirement of written informed consent for participation. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.SX: conceptualization, data curation, methodology, and writing—original draft. ZXB: data curation, methodology, resources, and writing—review and editing. YML: writing—review and editing. BL: resources and writing—review and editing. FLK: writing—review and editing. JZP: writing—review and editing. XGL: validation, supervision, and writing—review and editing. All authors contributed to the article and approved the submitted version.This work was funded by the Clinical and Translational Medical Research Fund, Chinese Academy of Medical Sciences (no. 2020-I2M-C&T-A-021). Funding source had no involvement in the conduct of the research and preparation of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Introduction: The risk of mortality is higher in pediatric intensive care units (PICU). To prevent mortality in critically ill infants, optimal clinical management and risk stratification are required.Aims and Objectives: To assess the accuracy of PELOD-2, PIM-3, and PRISM-III/IV scores to predict outcomes in pediatric patients.Results: A total of 29 studies were included for quantitative synthesis in meta-analysis. PRISM-III/IV scoring showed pooled sensitivity of 0.78; 95% CI: 0.72–0.83 and pooled specificity of 0.75; 95% CI: 0.68–0.81 with 84% discrimination performance . In the case of PIM-3, pooled sensivity 0.75; 95% CI 0.71–0.79 and pooled specificity 0.76; 95% CI 0.73–0.79 were observed with good discrimination power . PELOD-2 scoring system had pooled sensitivity of 0.78 (95% CI: 0.71–0.83) and combined specificity of 0.75 (95% CI: 0.68–0.81), as well as good discriminating ability  for mortality prediction in PICU patients.Conclusion: PRISM-III/IV, PIM-3, and PELOD-2 had good performance for mortality prediction in PICU but with low to moderate certainty of evidence. More well-designed studies are needed for the validation of the study results. The main aim of the pediatric intensive care unit (PICU) is to decrease mortality in infants by both monitoring and treating critically ill patients who are considered at risk of dying. To provide the better quality of care with available resources and optimal management of such patients, a suitable management plan and prioritization of resource utility after the identification of “at-risk” patients are needed . In ChinThe scoring system aims to predict the outcome during treatment and to provide a better quality of care with available resources. Many mortality prediction scoring systems are being used for predicting outcomes in PICU patients. Although it is a complicated process to assess the individual patient outcome precisely, there have been efforts to develop and validate models for prediction accuracy of outcomes, such as Pediatric Risk of Mortality (PRISM) III/IV, Pediatric index of mortality PIM-3), and Pediatric Logistic Organ Dysfunction-2 (PELOD-2). However, their predictive accuracy varied significantly in different populations worldwide , and Ped.The PIM was developed from data collected from PICUs in three prospective studies, from 1988 to 1995, and a cohort study, conducted from 1994 to 1997 by Shann et al. . PIM conPRISM score is another widely used model that was developed using data collected from PICUs in the United States. PRISM was later updated to PRISM-III and PRISM-IV with better calibration and discrimination efficiency  and is up = 0.317)  .Several recent studies have evaluated various prediction models to predict outcomes in PICU patients but have shown inconsistent findings, such as underestimation or overestimation of mortality prediction, poor discriminatory power, and absence of reporting of calibration statistics. , 11 As oStudy Design: Systematic review and meta-analysisEthical Clearance: Not Required.The present meta-analysis was conducted according to the reporting guidelines suggested in the PRISMA 2020 and Cochrane library. Search engines and electronic databases, such as Google Scholar, PubMed, and CENTRAL  were used to retrieve English language papers published up to May 2021. Free text words and medical subject heading (MeSH) terms were used, and the reference lists of potentially eligible studies and relevant review articles on a similar topic were scanned for additional possible studies. The following search key words were used: (((“pediatrics”[All Fields] OR “pediatrics”[MeSH Terms] OR “pediatrics”[All Fields] OR “pediatric”[All Fields] OR “pediatric”[All Fields]) AND ) OR “Pediatric Logistic Organ Dysfunction-2” OR “PELOD”[All Fields] OR “Pediatric index of mortality” OR “PIM”[All Fields]) AND ((“intensive care units”[MeSH Terms] OR (“intensive”[All Fields] AND “care”[All Fields] AND “units”[All Fields]) OR “intensive care units”[All Fields] OR “icu”[All Fields]) AND (“patient s”[All Fields] OR “patients”[MeSH Terms] OR “patients”[All Fields] OR “patient”[All Fields] OR “patients s”[All Fields])) AND . A search was restricted to human subjects only. The year of publication filter was 1996 and after.We included studies on patients admitted to PICU for any conditions.Studies with PRISM-III/IV, PIM-3, and PELOD-2 modelComparator: Threshold values reported in the published articlesOutcome: The outcome assessed was mortality. Mortality was defined as death at hospital or follow-up.Study design: All studies evaluating the accuracy of PELOD-2, PIM-3, or PRISM-III/IV scores to predict outcomes in pediatric patients admitted to the ICU. These prognostic models should aim to predict mortality at any time point in PICU patients aged <18 years.Not reporting relevant outcome  in PICU patients, case reports, review articles.Two independent authors screened the title, shortlisted the relevant articles, and extracted the data from the potentially eligible articles that meet the inclusion criteria of the study. Disagreements were resolved by discussion. The data extraction form consisted of the following information: first author of the published article, publication year, details of participants, sample size, details of prediction scoring system, settings, and country from where the data were reported.I2 statistic. The I2 = 50% was considered as significant heterogeneity. The methodological quality of studies was assessed using the PROBAST (Prediction Model Risk of Bias Assessment Tool) , and summary area under the curve with 95% CI. Heterogeneity was calculated with the nt Tool)  on four An adapted GRADE framework for determining the certainty of evidence in predictive accuracy studies was used . The GRAStudy characteristics are shown in A total of 18 studies reported sufficient data to compute pooled sensitivity and pooled specificity for the PRISM-III/IV scoring system. Sixteen studies were conducted in PRISM-III and two studies used PRISM-IV models. The meta-analysis of combined PRISM-III/IV studies showed pooled sensitivity of 0.78, 95% CI: 0.72–0.83, and a pooled specificity of 0.75, 95% CI: 0.68–0.81 . Our poop < 0.001) and specificity (p < 0.001) analyses (p = 0.81)  . We coulanalyses , with no = 0.81) . We obse = 0.81) . Our met = 0.81) . The lev = 0.81) .p = 0.14, I2 = 32.85), but significant heterogeneity was noted in pooled specificity  (p = 0.36)  and combined specificity of 0.76 (95% CI: 0.73–0.79) . No sign2 = 91%) . Publica = 0.36) . The sum = 0.36) . Our poo = 0.36) . The met = 0.36) . The cer = 0.36) .p < 0.001, I2 = 65.53% for sensitivity and 92.3% for specificity). Discriminatory performance was observed good as depicted by SROC 0.83; 95% CI 0.80–0.86 (p = 0.07)  . Our poo = 0.07) . Our met = 0.07) .In this study, we investigated the predictive accuracy and discriminating power of commonly used scoring systems such as PRISM-III/IV, PELOD-2, and PIM-3 to predict mortality risk in patients admitted to PICU. In China, mortality rates associated with PICU admission are approximately two or three times higher than in America and Europe. It is a need of the hour to identify predictor or prediction models of death in the PICU. There are constant explorations of death risk prediction score for providing optimal management to PICU patients with available resources.Accurate and reliable information about predicted mortality improves communication with patients about possible prognoses and optimal stratification of patients at risk. These three scoring systems have potential to provide the predictive accuracy for prognosis in PICU patients.We observed the evidence for good performance of these models; however, risk of bias assessment showed that evidence is with moderate to high risk of bias among studies. This bias was observed mainly due to inadequate presentation and reporting of statistical analysis, and failure to conduct the internal and external validation of models. The calibration of models is an essential component for evaluation of a test model; however, in our analysis, a total of 36% for PRISM-III/IV, 33% for PELOD-2, and 9% for PIM-3 models did not report the calibration of the model, which leads to bias in the statistical analysis domain. In the case of event per variable, 68% of studies in PRISM-III/IV, 88% of studies in PELOD-2, and 72% of studies in PIM-3 had <100 death events, resulting in high risk of bias as per PROBAST tool, which resulted into a risk of over fitting of the model in the validation studies. The most commonly used method to report calibration was the Hosmer–Lemeshow test, whereas this test is limited by neither the presence nor the magnitude of miscalibration . To overThe development of valid and reliable models for predicting mortality in PICU patients is an ongoing practice. We noted that the PRISM-III/IV score had the best predictive accuracy and discrimination in an individual patient (sROC 0.84), closely followed by PELOD-2 and PIM-3. We found the almost similar discriminatory performances of these scoring systems.Each of the prediction scores is applied at a specific timeframe in which reliable and optimal performance of prediction is to be expected. In the case of PRISM-III/IV scores, the optimal time point for prediction is after 24 h, while PIM-3 scores show the best performance and discrimination during the early hour after admission. A delayed timeframe that occurs in the case of PRISM-III/IV carries a risk of a patient dying before the assessment of PRISM-III/IV score, which could provide the probability of prognosis . On the p = 0.76. Good Calibration and discrimination of PELOD-2 were also reported in the study by Deshmukh et al.  published by Christoper et al.  conducteOur meta-regression analysis was to explore the source of variation on the discriminatory and predictive performance indicating the need of well-designed studies with additional clinically relevant variables to explore the source of heterogeneity between the studies.Regarding the certainty of evidence using GRADE analysis, we rated our certainty of evidence at very low for PRISM-III/IV, low for PELOD-2, and moderate for PIM-3 for predicting mortality in PICU patients. This means that the true effects are likely to be close to the estimated prognostic significance, but there are possibilities that it is substantially different.This study has several limitations. A high degree of heterogeneity was noted in the pooled analysis, which can originate from differences between study population, setting, and methodological quality of the studies. Considering the heterogeneity across the studies, further research will be necessary to obtain homogenous findings. A large sample size study reported by Christoper et al. could not be included in the analysis due to insufficient required data that resulted in the underestimation or overestimation of some of the studied scoring systems. Studies included in the meta-analysis were conducted in a wide range of conditions and settings leading to heterogeneity in the study findings.PIM-3, PELOD-2, and PRISM III/IV demonstrated good discriminatory power for mortality prediction in PICU patients with low to moderate quality of evidence. Further better-designed studies are needed to provide a better and accurate judgment of the performances of these models.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.YS and JJ conceived and designed the study, involved in literature search, data collection, and reviewed and edited the manuscript. YS analyzed the data. JJ wrote the paper. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


One-year later, alexithymic women still had significantly lowered in vitro production of IL-2 and IL-4, with lowered IL-2/IL-10 ratio and a reduced percentage of CD4. This is the first ever published study assessing cytokine production during a follow-up of alexithymics. Although our results should be interpreted with caution due the small sample size, they suggest a sustained reduction in both major type 1 and type 2 cytokines while the former seems to be more affected. The potential long-term health impact, if any, is still to be determined.Although previous cross-sectional studies suggested significantly dysregulated immune response in alexithymia, there is a lack of longitudinal studies. We sought to determine the reliability of the reported relationship between alexithymia and decreased immune response in a longitudinal study. Thirty-eight healthy women who had participated in a cross-sectional study were recontacted 1-year later. Of this sample, 26 were finally included: 13 females who had been found to be alexithymic, and 13 females who were classified as non-alexithymic under the 20-item Toronto Alexithymia Scale during the first phase of the study. A year later, they were still healthy women without any psychiatric disorders, their ages now ranging from 19 to 28 years old. Lymphocyte subset counts ,  Alexithymia is a clinical description coined by Sifneos  in 1972 In a cross-sectional study  comparinThe study sample was drawn from the same female university students as in the cross-sectional study , and, asFor this 1-year longitudinal study, we recalled only those who had measurements of cytokine production, i.e., the 38 healthy young women  who were recruited during the research carried out in 2004–2005 . A year The absence of a current/past major depressive episode, anxiety disorders or any other psychiatric disorders according to DSM-IV was assessed again, using the M.I.N.I. , 30. It After an overnight fast, blood samples were collected in heparinized tubes between 8:00 a.m. and 9:00 a.m. and immediately transported to the laboratory. To determinate the WBC subset counts, the total number of WBC and leukocyte differential counts were calculated with a Coulter counter . Enumeration by flow cytometry with FACS count (Becton Dickinson) included the following cells: two types of T cell subsets CD4, CD8.Serum levels of cortisol were measured using a competitive enzyme immunoassay which was performed entirely within the ST AIA-PACK CORT (Tosoh Bioscience).2 humid incubator, in the presence of phytohemagglutinin A  at a final concentration of 2.5 μg/ml; for 48 h. All cultures were performed in RPMI 1640 with Glutamax (Gibco Life sciences) supplemented with 1% sodium bicarbonate, 1% penicillin streptomycin (both from Gibco), and 25 μl/500 ml mercaptoethanol (Sigma). This culture medium was itself supplemented with 10% heat-inactivated FCS. At the end of this period, culture supernatants were harvested by lymphocyte centrifugation and kept at −20° until ELISA assay. Commercial kits  were used for all ELISA assays. Samples were tested in duplicate in the same assay. Inter- and intra-assay variabilities were always <10%. The lower limit of detectability was 1 pg/ml for IL-1β, 7 pg/ml for IL-2, 0.03–0.22 pg/ml for IL-4, 3.9 pg/ml for IL-10.Lymphocytes were separated on a 25 ml Ficoll layer, cushioned by a 5 ml RPMI 1640 culture medium. This was done by spinning for 20 min at 1,200 rpm (g) in a Hereaeus centrifuge with swing out buckets at 4°C. Lymphocytes which were collected at the interface were washed twice in culture medium before being counted for viability using the Trypan blue dye exclusion test. Viability was usually well above 90%. 5 × 10 cells /ml were then cultured in 25 ml ventilated culture flasks  in a Fetal Calf Serum (FCS) supplemented culture medium, at 37°C in a 5% COt-tests were used. In the absence of normal distributions non-parametric tests (Mann and Whitney Wilcoxon) were performed. Most parameters were normally distributed with the exception of cytokine production. Differences in cell counts and serum concentrations of cortisol were determined using t-tests; for cytokine production, Mann and Whitney Wilcoxon tests were used according to the distribution. The results are presented as mean ± standard deviation. The effect size r was calculated for Wilcoxon tests (case of independent two-samples test), and Cohen's d for t-tests. The r-value varies from 0 to close to 1. The interpretation values for r commonly in published litterature and on the internet are: 0.10– < 0.3 , 0.30– < 0.5 (moderate effect), and ≥0.5 (large effect).Data were analyzed using SPSS (27.00) software. The data were examined for normal distributions with the Shapiro-Wilk test. For normal distributions, There were no differences in the sociodemographic and psychopathological characteristics  for the subjects lost to follow-up compared to the 26 subjects who were included both in the cross-sectional study and 1-year later in the longitudinal study.n = 13) and non-alexithymics (n = 13) regarding age  and none of the subjects were suffering from tobacco addiction according to the Fagerström test. What is worthy of note is that there were no longer any differences (see p = 0.28) or the HADS subscale for depression .One-year later, there was still no difference between alexithymics  1-year later. On the other hand, although there was no longer any significant difference, there was a tendency toward decreased CD4/CD8 levels in alexithymic subjects (p = 0.1). We did not observe, 1-year later, any significant differences in terms of the percentage of lymphocytes or the neutrophil-to-lymphocyte ratio . In addition, in comparison with the non-alexithymics, alexithymics still had lowered IL-2/IL-10 ratio (p = 0.037) but there was no longer any significant difference concerning in vitro production of IL-1β  and perhaps to the fact that this cytokine is less specific to the immune dysregulation observed in alexithymia.One-year later, we did not find any significant IL-1β To our knowledge, this is the first longitudinal study ever published assessing the immune parameters (notably cytokines) in alexithymics compared to non-alexythymics.n = 93) comparatively to non-alexithymic subjects (n = 74). These results combined with ours suggest a decreased immune response in subjects afflicted with alexithymia.Temoshok et al.  found, iA brief review of the literature shows that most studies evaluating immunity in alexithymics are based on immunocyte counts or on the measurement of circulating levels of cytokines. Several other authors found decreased immunity in alexithymics in cross-sectional studies.Todarello et al. , 22 and n = 22) compared to non-alexithymic (n = 37) patients undergoing upper endoscopy, regardless of stress, anxiety, and depression. In 24 male and female patients suffering from Somatoform Disorder (SFD), Pedrosa Gil et al.  at least 1-year later. Obviously, we need more longitudinal studies in this area of research, with larger samples, to better understand the possible immune dysfunction in alexithymic subjects and its potential long-term health impact notably the risk of infectious diseases and immunological disorders as seen under chronic stress conditions , 51, 54.The original contributions presented in the study are included in the article/supplementary files, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Paris-Hospital Hotel Dieu Ethics Committee. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This article is dedicated to the memories of Claire Perrin and Gerard Chaouat.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Targeted therapy refers to exploiting the specific therapeutic drugs against the pathogenic molecules (a protein or a gene) or cells. The drug specifically binds to disease-causing molecules or cells without affecting normal tissue, thus enabling personalized and precision treatment. Initially, therapeutic drugs included antibodies and small molecules, (e.g. nucleic acid drugs). With the advancement of the biology technology and immunotherapy, the gene editing and cell editing techniques are utilized for the disease treatment. Currently, targeted therapies applied to treat cardiovascular diseases (CVDs) mainly include protein drugs, gene editing technologies, nucleic acid drugs and cell therapy. Although targeted therapy has demonstrated excellent efficacy in pre-clinical and clinical trials, several limitations need to be recognized and overcome in clinical application, . This review introduces the mechanisms of different targeted therapies, and mainly describes the targeted therapy applied in the CVDs. Furthermore, we made comparative analysis to clarify the advantages and disadvantages of different targeted therapies. This overview is expected to provide a new concept to the treatment of the CVDs. Cardiovascular diseases are the leading cause of death worldwide . CVDs arThe success of the human genome project and the rapid development of molecular biology facilitate the precise detection of genome, transcriptome and proteome changes. By using these methods, researchers can explore the mechanisms underlying the progression of diseases and design new drugs that target to the pathogenic molecules, which is named targeted therapy . By specMYH6 in hypertrophic cardiomyopathy (HCM)) )  or an aby (HCM)) . ActuallAntibodies can specifically recognize and bind to the epitopes of the antigen. Based on the number of binding epitopes, antibodies used for targeted therapies can be classified as mAbs or bispecific antibodies (bAbs). Here, we summarize the mechanisms and applications of the two types of antibodies.At present, mAbs have been widely applied in malignancies and rheumatic diseases. And mAbs have been exploited to treat CVDs . mAbs exp.S127R, p. F216L and p. D374Y) of proprotein convertase subtilisin/kexin type 9 (PCSK9) can lead to autosomal dominant hypercholesterolemia (by reducing low-density lipoprotein receptor (LDLR) levels) and increase the susceptibility to CVDs  or placebo . During the study, the dose of alirocumab was adjusted to maintain the cholesterol level at 0.65–1.30 mmol/L. The primary endpoint was a composite of death from fatal or nonfatal ischemic stroke, nonfatal myocardial infarction (MI), coronary heart disease (CHD), or unstable angina (UA) requiring hospitalization. After 2.8 years of follow-up, the results showed that the alirocumab could reduce the incidence of the primary endpoint . Patients with a higher baseline LDL-C level(> 100 mg/dl) gained more benefits than patients with a lower baseline LDL-C level. Besides, evolocumab is another mAb that inhibits PCSK9. Binding of the evolocumab to PCSK9 resulted in a rise in hepatic surface LDLR and an increase in plasma LDL-C clearance (p = 0.0008) and without  diabetes. However, the long-term efficacy of the evolocumab on high LDL-C needs to be evaluated in further studies with longer follow-up.Possessing the above characteristics, mAbs have also shown excellent efficacy in the cardiovascular field. Gain-of-function mutations  or placebo (n = 22) (Angiopoietin-like protein 3 (ANGPTL3) can inhibit the activity of lipoprotein lipase and increase the content of triglyceride and other lipids in plasma . Loss-ofk of CHD . Accordik of CHD . In a ph(n = 22) . All patn = 2,263), 150 , 300 mg  or placebo , given subcutaneously once every 3 months. During a median follow-up of 3.7 years, 385 patients had the HHF event. Compared with the placebo group, the unadjusted HRs for HHF in different dose group were 1.04  for 50 mg, 0.86  for 150 mg, and 0.76  for 300 mg (p = 0.025). Canakinumab could reduce the incidence of HHF in a dose-dependent manner (n = 15), canakinumab 300 mg (n = 15) or placebo (n = 15). The primary endpoint was the time in days from randomization to either discharge from the hospital or an improvement of two points on a seven category ordinal scale. The trial is still in progress. Rilonacept is a soluble IL-1 decoy antibody. Two IL-1 receptor chains extend to the out space of the membrane, fusing to form a “trap” for neutralizing IL-1α or IL-1β  has been indicated to contribute to CVDs via mediating the inflammatory response, and act as an upstream regulator of a series of inflammatory cytokines . Based ot manner . Canakint manner . A totalor IL-1β . In 2008or IL-1β . At presor IL-1β . The trior IL-1β .n = 58) or placebo (n = 59) before coronary angiography. The primary endpoint was defined as the changes of hsCRP. The results showed that hsCRP in the placebo group was 2.1 times higher than that of tocilizumab . High sensitivity troponin T in the placebo group was 1.5 times higher than that in the tocilizumab group (234 vs. 159 g/L/h). After 6 months of follow-up, no safety events were detected. The results indicated that tocilizumab was sufficient to lessen the inflammatory response and myocardial injury in NSTEMI patients.Interleukin-6 (IL-6) has been reported to cause the development and instability of arterial plaques , particiAbciximab, a Fab fragment of chimeric human-mouse mAb 7E3 , could bMAbs can also effectively treat diseases caused by certain abnormal proteins with high specificity. For instance, abciximab targeting to (GP) IIb/IIIa to treat STEMI has shown a good therapeutic effect in the clinic . Given tGenerally, the occurrence and development of the diseases may be driven by different molecules, in which the monotherapy of mAbs is insufficient for treatment and combined therapy is required . HoweverMET proto-oncogene, and develop drug resistance  Bridsistance . Herein,sistance . 3) Cofasistance . 4) Piggsistance .n = 9) or phosphate-buffered saline . Increased HSCs number in the damaged area and improved cardiac function were observed in animals treated with the bAbs . In vivo, GPVI-CD133 bAb enhanced the reendothelialization in mice with the carotid injury. But mice treated by GPVI mAb + CD133 mAb did not show any elevation of EPCs. Therefore, the GPVI-CD133 bAb is a promising treatment for vascular lesions. These results indicated that bAbs could simultaneously bind to two epitopes and realize a “1 + 1 > 2” therapeutic effect.In addition to ischemic cardiomyopathy, bAbs are also used to treat vascular lesions. The integrity of the endothelial cells (ECs) plays an essential role in the cardiovascular system. When the integrity of ECs is destroyed, the subendothelial matrix proteins will expose, causing the formation of the platelet-mediated thrombus. GPVI is a soluble platelet collagen receptor . CD133 iIn the present, peptides are utilized to treat hypertension and vascular diseases. ATRQβ-001, a peptide vaccine made of human angiotensin II (AngII) receptor I and Qβ phage virus-like particles, can inhibit the Ang II-mediated pathway, thus being applied in the treatment of hypertension and aneurysms. In Liao’s et al. studies, the ATRQβ-001 vaccine could effectively decrease the blood pressure in the AngII induced hypertension mice and spontaneous hypertensive rats (SHRs) . The teaATR12181, the extracellular part of angiotensin receptor 1A (AT1A), is also used to treat SHRs . After rIn vitro, the anti-ETR-002 antibodies could attenuate the Ca2+-dependent signal transduction induced by endothelin-1. In vivo, the ETRQβ-002 vaccine significantly decreased the right ventricular systolic pressure by 10 mmHg in Sugen/hypoxia-induced PAH mice and 20 mmHg in monocrotaline-induced PAH rats. No significant immune injury was observed in the vaccinated animals.Liao’s team also developed the first vaccine against endothelin-1 receptor type A (ETAR) (ETRQβ-002 vaccine) to treat the pulmonary arterial hypertension (PAH) . In monoIt is worth mentioning that although animal experiments have shown positive effects, clinical trials are needed to test the safety and efficacy of these vaccines.6 IU) or placebo once daily for 5 days. The study is expected to provide evidence to the immunomodulatory treatment via cytokines in the CVDs.Besides using mAbs to antagonize the pro-inflammatory cytokines, researchers also used some cytokines with immunomodulatory function to treat diseases. Regulatory T cells (Tregs) can maintain immune homeostasis and play a crucial role in myocardial repair and atherosclerosis . InterleCRISPR/Cas9 is associated with immunity against the virus and foreign DNA . It recoMYBPC3 mutation and a CRISPR/Cas9 system resulted in a 100% correction of the mutation.HCM is mainly caused by autosomal dominant inheritance, and there are 1,500 mutations in 15 genes known to cause the disease . For fam2+ homeostasis. The enhanced function of the PLN will impair the heart function  participates in the process of the Cafunction . Kaneko SPR/Cas9 . Comparelesterol . Moreoveertrophy . After bertrophy .TTR gene mutation and characterized by excessive deposition of thyroxine in the myocardium  to explore the feasibility of CRISPR/Cas9 for the treatment of DMD (Thyroxine (TTR) amyloidosis is caused by the ocardium . Finn etat model . Howeverat model . DMD cant of DMD . Resultst of DMD . CRISPR/MYL3 gene (a cardiomyopathy-associated genetic variant) (p.Ala57Asp) was shown to be likely pathogenic in the Clinvar database. Patient-derived iPSCs were edited by CRISPR/Cas9 to generate four iPSC lines: 1) “healthy” control without any mutations; 2) homozygous MYL3 VUS (170C > A); 3) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C > G); and 4) heterozygous MYL3 frameshift mutation (170C > A/fs). Only cell lines carrying the known heterozygous MYL3 pathogenic mutation showed an HCM phenotype at the morphology, gene expression, or functional levels. The above results illustrated the ability of CRISPR/Cas9 to discriminate between benign and pathogenic mutations, thus providing guidance for clinical risk assessment and therapeutic intervention. Other studies showed that CRISPR/Cas9 could be used to identify the VUS that contributed to the CVDs  is gene mutation for which the pathogenicity is not clear. Ma et al. used CRISPR/Cas9 to analyze an asymptomatic individual carrying mutations in the variant) . The mutthe CVDs .ANGPTL3 is associated with lower risk of CHD. Musunuru et al. screened the potential gene editing sites using Neuro-2a cells and selected the codon Gln-135 site with the protospacer sequence AGCCCTTCAACACAAGGTCA on the ANGPTL3 gene (Pcsk9 Trp-159 (BE-Pcsk9), with sgRNA targeting to Angptl3 Gln-135 (BE-Angptl3). Primarily, BE-control and BE-Angptl3 were injected into the C57BL/6J mice. On the day 7, deep sequencing of the liver samples revealed a median editing rate of 35% in the BE-Angptl3 group, while no gene editing was observed in the BE-control group. Notably, deep sequencing of the top 10 predicted off-target sites demonstrated no evidence of gene editing. Baseline levels of triglycerides, cholesterol, and plasma ANGPTL3 were comparable between the groups, whereas on day 7, significantly lower levels of triglycerides, cholesterol and plasma ANGPTL3 were observed in the BE-Angptl3 group. Next, the mice were treated with BE-control, BE-Pcsk9, BE-Angptl3, or a 1:1 mix of BE-Pcsk9 and BE-Angptl3. On day 7, the triglycerides of the BE-Angptl3 group showed a greater reduction than the BE-Pcsk9 group. Neither synergism nor additivity was observed in the BE-Pcsk9 + BE-Angptl3 group. Finally, the researcher used the Ldlr-knockout mice to test the effect of the BE-Angptl3. On day 14, compared with the BE-control, BE-Angptl3 markedly reduced the level of cholesterol (51%) and triglycerides (56%) in treated mice.BE is a single base editing technology, which can cause the change from cytosine to thymine using the Cas protein at a specific site . InitialTL3 gene . Mice weFBN1 gene can lead to the Marfan syndrome . Results showed that 10 of the 20 clones (50%) were edited, and eight clones presented a perfect C to T correction. Subsequently, the experiment was carried out in human embryos. In the testing group, seven embryos were treated by BE and correctional sgRNA. The control group consisted of seven embryos treated by BE and the scrambled sgRNA. Sanger sequencing demonstrated a 100% correction in the testing group.Heterozygous T7498C mutation of the syndrome . Huang esyndrome . Cells cCAM) can cause long QT syndrome (LQTs)  or inhibit (CRISPRi) the transcription of the target genes . The case (LQTs) . The CAMmutation . The iPSin vivo and in vitro experiments concluded that a careful design of the sgRNA could avoid the off-target events  and transcription activator-like effector nucleases technology (TALENs), which consist of the DNA recognition domain and DNA splicing domain, can also be considered as a way for gene editing. ZFN and TALENs have been successfully applied to correct mitochondrial DNA mutations . Accordi of mice .The nucleic acid drugs mainly include DNA, microRNA (miRNA), small interfering RNA (siRNA), antisense oligonucleotides (ASO), and mRNA .On the one hand, part of viruses, such as retrovirus, lentivirus, adenovirus and adeno-associated virus (AAV) are commonly used as vectors for nucleic acid drugs . Among tAAV can transfect the cells via the glycosylated cell surface receptors. Following the transfection, AAV will be either transported into the nucleus or degraded by the proteasome. There are two kinds of recombinant AAV: single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) . The ssALAMP2b gene, the concentration of LAMP2b protein in heart, liver and skeletal muscle tissue of the LAMP2 knockout mice notably retrieved, and the symptoms were improved in a dose-dependent manner  is a rare X-linked autophagic vacuolar myopathy, which is characterized by multiple system abnormalities, such as heart, skeletal muscle, and liver . The pent manner . The abo2+-ATPase (SERCA2a) plays a crucial role in the process of Ca2+ homeostasis. Besides, decreased activity and expression of SERCA2a were reported in HF patients (n = 127) or AAV1/SERCA2a (n = 123) (The sarcoplasmic reticulum Capatients . In CUPIn = 123) . The priProinflammatory cytokines constitute a key part in the pathogenesis of HF . RegnaseHypertension can lead to diastolic dysfunction, cardiac remodeling and fibrosis . BNP canin vivo experiments showed that overexpression of miR-25 reduced cardiac function in TAC mouse models   Wilson . Inversee models . The TACe models . These rogenesis . In MI mogenesis . To furtogenesis .Currently, a line of preclinical studies focusing on the miRNA therapy are ongoing . It shouThe siRNA refers to an exogenous double-strand RNA . After dThe ASO can combine with mRNA and inhibit gene expression via the following two manners: 1) RNaseH independent way: After combining with the complementary mRNA, the ASO inhibits translation via the steric blocking effect. 2) RNaseH dependent way: After combining with the complementary mRNA, the ASO recruits the RNaseH to degrade the mRNA .n = 33) or placebo group (n = 33)  via the RNaseH dependent way, thus decreasing the levels of LDL-C, TC and non-HDL-C in patients with familial hypercholesterolemia . Familia(n = 33) . The pri(n = 33) . These dThe mRNA can be directly translated into proteins. At present, the application of mRNA therapy includes two aspects: 1) Introducing exogenous mRNA into the body and correcting the deficiency of gene expression. 2) Loading the mRNA into the vaccine.2. The primary endpoint was to evaluate the tolerability and safety of the modRNA. Compared with the control group , the VEGF-A protein level in the modRNA group was significantly higher. Laser Doppler measurement and acetylcholine iontophoresis suggested that the skin blood flow of the modRNA group increased 2 times. These results indicated that modRNA encoding the VEGF-A could promote vasodilation and neovascularization in T2DM patients. This study supported evidence for the potential that mRNA can effectively treat ischemic symptoms in patients with ischemic CVD and T2DM.To date, some mRNAs have been developed to treat CVDs. Chien et al. successfully treated the MI mice by using chemically modified mRNA (modRNA) encoding VEGF-A . Based oThese results show that the mRNA plays an important role in the diseases caused by loss-of-function mutation.The CAR-T therapy belongs to immune therapy. The CAR is composed of three parts: 1) An antigen binding region, which consists of a single-chain fragment variable (scFv) and can specifically bind to the target antigens; 2) The transmembrane area, which fixs the scFv on the surface of T cells; 3) Signal transduction region, which consists of CD3-ζ chain of the T cell. When the scFv binds to the target antigen, the CAR-T cells will be activated via the major histocompatibility complex-independent way.  Depil e.The CAR-T therapy has successfully treated myocardial fibrosis in mice . The ovaTargeted therapy is a promising method to achieve the precision medicine. Nowadays, various technologies accelerate the development of the targeted therapy. The main technologies of the targeted therapy include protein drugs, gene editing technology, nucleic acid drugs and cell therapy .The protein drugs consist of mAbs, bAbs and peptides. At first, the mAb is generated from the mouse hybridoma, which carries the heteroantigens and induces the human anti-mouse antibody response . The sidMYBPC3 mutation and a CRISPR/Cas9 system resulted in a 100% correction of the mutation, indicating the excellent efficiency of CRISPR/Cas9  and drugs that inhibit gene expression . The delivery of the nucleic acid drugs into the host necessitates a vector, and the AAV is the most commonly used carrier . For disin vitro, the CAR-T cells can specifically kill the target cells without the MHC restriction (After edited triction . Epsteintriction . Given ttriction .In conclusion, targeted therapy has emerged as a novel and promising approach for the treatment of CVDs . By util


N-hydroxysuccinimide (NHS) as crosslinking agents. The synthesis products were characterized using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and thermogravimetric analysis (TGA). Thermogravimetric analysis showed that CBX-MG and CBX-MG-PEI presented a lower decomposition temperature than MG. The CBX-MG-PEI has a high buffer capacity in the pH range of 4 to 7, similar to the b-PEI. In addition, the CBX-MG-PEI showed an improvement in hemocompatibility in comparison with the b-PEI. The results showed a non-hemolytic property at doses lower than 0.1 µg/mL (CBX-MG-PEI). These results allow us to propose that this copolymer be used in transfection, polymeric nanoparticles, and biomaterials due to its physicochemical and hemocompatibility properties.In the present study, the modification of branched polyethyleneimine (b-PEI) was carried out using mesquite gum (MG) to improve its hemocompatibility to be used in biomedical applications. In the copolymer synthesis process (carboxymethylated mesquite gum grafted polyethyleneimine copolymer (CBX-MG-PEI), an MG carboxymethylation reaction was initially carried out (carboxymethylated mesquite gum (CBX-MG). Subsequently, the functionalization between CBX-MG and b-PEI was carried out using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and  Branched polyethyleneimine (b-PEI) is one of the most widely used synthetic polycations because of its chemical functionality arising from the high density of amines . This poPEI/DNA polyplexes in the bloodstream . Fi. Fi44]. N-hydroxysuccinimide (NHS)  were added to the solution at a molar ratio of 1:4:4 (CBX-MG: EDC: NHS), and the reaction mixture was stirred at room temperature for 24 h to activate the carboxyl group of CBX-MG. Next, 15 g of b-PEI  was dissolved in water, and it was added to the activated CBX-MG solution. Then, the pH of the solution was maintained at 6.5 and the reaction continued for another 48 h at room temperature. Finally, the resultant solution was purified by exhaustive dialysis against deionized water for 24 h, followed by lyophilization to obtain CBX-MG-PEI.Carboxymethylated mesquite gum grafted polyethyleneimine copolymer (CBX-MG-PEI) was synthesized by adapting the previously reported method by Jana et al.  22]. Fi. Fi22]. FTIR analysis was carried out using a Thermo Nicolet 6700 FTIR . XPS analysis was carried out using an XPS Escalab 250Xi . Thermogravimetric analysis was carried out using a Q600 SDT thermal analyzer  at a heating rate of 10 °C/min, ranging from room temperature to 800 °C, with a flow of nitrogen gas (purity 5.0) at 100 mL/min. The determination of potential zeta was carried out by a Zetasizer Nano ZS . The absorbance measurements of the hemolysis tests were taken using the NanoDrop ND-1000 UV-Vis Spectrophotometer .A semiquantitative analysis through FTIR spectroscopy was carried out. For this analysis, an appropriate baseline in the CBX-GM-PEI spectra was determined by using Origin software, and a Gaussian iterative curve fitting of the deconvoluted peaks afforded a means of evaluating the areas of each peak. The percentage of conjugation of b-PEI with CBX-MG was based on the peak area ratios of amide I/amine I band  and calcamide I: Area of the peak associated with amide I (1640.8 cm−1)AAmine I: Area of the peak associated with amine I (3420 cm−1)AOrigin software, and a Gaussian iterative curve fitting of the deconvoluted peaks afforded a means of evaluating the areas of each peak. The percentage of conjugation of b-PEI with CBX-MG was based on the peak area ratios of Aamide/Atotal area and calculated through the following equation:A semiquantitative analysis through XPS spectroscopy was carried out. For this analysis, an appropriate baseline in the CBX-MG-PEI spectra was determined by using amide: Area of the peak associated with amide in the N1s spectra (399.8 cm−1)Atotal area: Sum of the areas in the N1s spectraAThe CBX-MG-PEI polymer solution was prepared in a 50 mL flask , and pure water was used as a control. After adjusting the initial pH to 10.0 with 0.1 M NaOH, 25 µL increments of 0.1 M HCl were titrated into the solution while measuring the pH response with a pH electrode. The recorded pH varied from 10.0 to 3.0 .A hemolysis test was developed to determine the effect of polymers on erythrocyte lysis. The protocol followed in the present work was based on the investigation reported by Roacho et al. with some modifications .A spray-coated EDTA tube  with blood from a healthy donor was used. Erythrocytes were isolated by centrifugation (3000 rpm for 4 min) and washed three times with phosphate-buffered saline (PBS). Polymers were dispersed in PBS (0.6–0.01 µg/mL). Quadrupled samples of polymers were incubated with 98 µL of an erythrocytes suspension (1:99 erythrocytes:PBS). The incubation conditions were 37 °C in agitation at 300 rpm for 30 min. The positive control was a suspension of erythrocytes in distilled water. The negative control was a suspension of erythrocytes in PBS. After the incubation, the samples were centrifuged , and the hemoglobin that was released in the supernatant was measured by ultraviolet–visible (UV–vis) spectroscopy at 415 nm. The positive control absorbance was a reference as 100% of hemolysis, and each of the samples was calculated according to the positive control. The average absorbance of the negative control was subtracted from each sample.−1 and 2922 cm−1, respectively. A band centered near 1600 cm−1 is assigned to amide I, attributed to the protein content of the samples (arising from the MG glycoprotein); COO− asymmetric stretching bands, located at 1416 cm−1, were also identified. The bands around 1011 cm−1 and 990 cm−1 can be attributed to vibration modes of the C–O and the C–O–H groups of carbohydrates . The band at 834 cm−1 indicates the occurrence of pyranose glycosidic acetal groups. According to Moreno-Trejo et al., these results were reported for the characterization of purified MG, and this material was used to synthesize silver nanoparticles and stabilize essential citrus oil nanoemulsions [−1, 1310cm−1, and 1060 cm−1, corresponding to the COO− asymmetric and symmetric stretching vibration due to the carboxyl group of the carboxymethyl moiety of CBX-MG. These results correlate with those obtained by other authors who have carried out carboxymethylation in other polysaccharides [FTIR spectroscopy was used to further confirm the chemical modification of b-PEI with MG through carbodiimide chemistry . The FTImulsions ,48. On tcharides ,50,51.−1 and 3280 cm−1) and the amide group  were evidenced. The assignments of these peaks can be observed in Regarding the results obtained for the CBX-MG-PEI, peaks associated with the primary amine , the absence of the signal associated with the O-C=O group was evidenced in the C1s spectrum , which iThese results confirmed that the CBX-MG-PEI copolymer was successfully synthesized through the formation of the amide bond between carboxyl groups of carboxymethylated CBX-MG and the amino groups in b-PEI.amide I/Aamine) [amide/Atotal area, respectively). The results obtained are shown in The percentage of conjugation of PEI with carboxymethylated mesquite gum was calculated through semiquantitative analysis through FTIR and XPS spectroscopy (N1s spectra) based on the calculation of the peak area ratios ((A/Aamine)  and AamiTaking into account the results shown in The TGA technique allows studying the decomposition pattern and thermal stability of materials. The results  and discThe zeta potential is an important parameter to examine the surface charge of the polymers, depending on the pH of the solution. CBX-MG-PEI showed higher positive zeta potential in the acidic pH region due to the protonation of the amino groups of b-PEI. On the other hand, at the higher pH range, CBX-MG-PEI showed less positive zeta potential value due to fewer positively charged ions . The resThe majority of cationic polymers have a high buffering capacity, disrupting endosomes during transfection, thereby facilitating the escape of the polymer/DNA complex ,58. FiguAssessment of Hemolytic Properties of Materials ASTM F756-08 were analyzed, which indicate that the hemolytic activity of the materials is classified in three types: non-hemolytic materials (0–2% of hemolysis), low hemolytic materials (2–5% of hemolysis), and high hemolytic materials (higher than 5% [A hemolysis assay is an essential initial step in evaluating the blood compatibility of polymers since this test can predict the potential side effects for polymeric vectors in intravenous administration . This as than 5% . For the than 5% . In cont than 5% . The imp than 5% ,23,65.This study developed a novel copolymer based on carboxymethylated mesquite gum grafted polyethyleneimine, characterized by FTIR, XPS, zeta potential, and TGA. The copolymer keeps an essential property of b-PEI, which is the buffer capacity. This property is critical for its application as a non-viral vector in gene therapy. Additionally, the results obtained in the hemolysis assays indicate that the introduction of MG in the structure of b-PEI decreases the hemotoxicity of the latter. This knowledge will open different copolymer applications, for example, as a non-viral vector in gene therapy against cancer. These results allow us to propose that this copolymer can be investigated in transfection studies, preparation of polymeric or metallic nanoparticles, and in general as a biomaterial due to its physicochemical and hemocompatibility properties.